Methods for treatment of diabetes using peptide analogues of insulin

ABSTRACT

The present invention is directed toward peptide analogues of insulin B chain that are generally derived from peptides comprising residues 9 to 23 of the native B chain sequence. The analogues are altered from the native sequence at position 12, 13, 15 and/or 16, and may be additionally be altered at position 19 and/or other positions. Pharmaceutical compositions containing these peptide analogues are provided. The peptide analogues are useful for treating and inhibiting the development of diabetes.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.10/339,160, filed Jan. 8, 2003, now U.S. Pat. No. 6,933,274, whichapplication is a continuation of Ser. No. 09/787,140, filed Jun. 7,2001, now U.S. Pat. No. 6,562,942, which is a National Stage Applicationof PCT/US99/03915 (35 U.S.C. 371), international filing date of Feb. 23,1999, which claims foreign priority benefits under 35 U.S.C. 119 fromU.S. application Ser. No. 09/028,156, filed Feb. 23, 1998, nowabandoned, all of which are incorporated herein by reference in theirentirety.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates generally to peptide analogues of insulin,and more specifically to methods for treating diabetes using peptideanalogues derived from residues 9-23 of human insulin B chain.

2. Description of the Related Art

Insulin dependent diabetes mellitus (IDDM) is an organ specificaautoimmune disease affecting close to a million people in different agegroups in the United States. The disease is characterized by extensivedestruction of the insulin producing beta cells in the pancreatic isletsand dysregulation of glucose metabolism leading to frank diabetes. Thedefining feature of IDDM is the lymphocytic infiltration of the islets.Among the invading cells, T cells appear to be one of the majormediators of autoimmune destruction.

Type I diabetes is further characterized by increased levels ofantibodies to various islet associated antigens, including insulin,GAD65, GAD67 and ICA512. These antibodies can be detected much beforefrank disease, and an immune response to such antigens can be used as apredictor for impending diabetes in patients with susceptible genetic(HLA) haplotypes.

Currently, patients are dependent on insulin injections to maintainnormoglycemia. Insulin is a polypeptide hormone consisting of twodisulfide-linked chains, an A chain consisting of 21 amino acid residuesand a B chain of 30 residues. While administration of insulin providessignificant benefits to patients suffering from diabetes, the shortserum half-life of insulin creates difficulties for maintaining properdosage. The use of insulin also can result in a variety of hypoglycemicside-effects and the generation of neutralizing antibodies.

In view of the problems associated with existing treatments of diabetes,there is a compelling need for improved treatments that are moreeffective and are not associated with such disadvantages. The presentinvention exploits the use of peptide analogues which antagonize a Tcell response to insulin to effectively treat diabetes, while furtherproviding other related advantages.

BRIEF SUMMARY OF THE INVENTION

The present invention provides compounds and methods for treating andpreventing diabetes. Within certain aspects, the present inventionprovides peptide analogues comprising residues 9 to 23 of human insulinB chain (SEQ ID NO:2), wherein the peptide analogue differs in sequencefrom native human insulin B chain residues 9 to 23 due to substitutionsat between 1 and 4 amino acid positions. Such substitutions may be madeat one or more residues selected from the group consisting of residues12, 13, 15 and 16, with or without additional substitutions at otherresidues. Within certain preferred embodiments, such substitutions mayoccur at two or three amino acid residues within residues 9 to 23 ofinsulin B chain. Substitutions may also occur at residue 19.Substitutions are preferably non-conservative, and analogues whereinresidue 12, 13, 15, 16 and/or 19 are altered (to, for example, alanine)are preferred. Analogues further comprising residue 24 of insulin Bchain are also preferred. In certain other embodiments, the peptideanalogues comprise no more than 18 residues, no more than 16 residues orno more than 15 residues of human insulin B chain.

Within further embodiments, the peptide analogues consist essentially ofresidues 9 to 23 or 9 to 24 of human insulin B chain (SEQ ID NO:2),wherein the peptide analogue differs in sequence from native humaninsulin B chain residues 9 to 23 due to substitutions at between 1 and 4amino acid positions, and wherein at least one substitution occurs at aresidue selected from the group consisting of residues 12, 13, 15 and16.

Within further aspects, pharmaceutical compositions are provided,comprising a peptide analogue as described above in combination with aphysiologically acceptable carrier or diluent.

The present invention further provides methods for treating and/orinhibiting the development of diabetes, comprising administering to apatient a therapeutically effective amount of a pharmaceuticalcomposition as described above.

These and other aspects of the invention will become evident uponreference to the following detailed description and attached drawings.In addition, various references are set forth below which describe inmore detail certain procedures or compositions. These references areincorporated herein by reference in their entirety as if each wereindividually noted for incorporation.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the amino acid sequence of residues 9-23 of insulin Bchain (SEQ ID NO:2).

FIG. 2 is a graph showing the proliferative response (measured in cpm)of a NOD mouse T cell clone to a native insulin B chain (9-23) peptidein the presence of varying amounts of representative peptide analogues,in which different residues are substituted with alanine, as indicated.

FIG. 3 is a graph showing the proliferative response (measured in cpm)of a NOD mouse T cell clone to a native insulin B chain (9-23) peptidein the presence of varying amounts of the representative peptideanalogue in which amino acids at positions 16 and 19 are substitutedwith alanine (NBI-6024; indicated by squares). For comparison theproliferative response in the presence of an unrelated control peptidederived from myelin basic protein (NBI-5096; indicated by circles) isalso shown. The response is shown as mean CPM±SEM of triplicatecultures.

FIGS. 4-6 are histograms illustrating the proliferative response(measured in cpm) of T cell lines from different diabetic patients tothe native B chain (9-23) peptide or to representative peptideanalogues. Peripheral blood mononuclear cells were isolated fromdiabetic patients and cultured in the presence of insulin B chain (9-23)peptide. After three rounds of restimulation with insulin B chain(9-23), 1×10⁵ T-cells and 7×10⁴ irradiated autologous PBMCs were addedto each well in a round bottom 96-well plate in complete medium. Cellswere cultured for 5 days with NBI-6024 (insulin B chain 9-23 withalanine substitutions at positions 16 and 19), insulin B chain (9-23) ormedium only. On day 4, the cells were pulsed with ³H-thymidine andre-cultured for an additional 18 hours. The cultures were thenharvested, counted using liquid scintillation, and the data wasexpressed as the mean counts per minute (cpm) of replicatesamples±standard error of the mean (sem).

FIG. 7 is a histogram illustrating the proliferative response (measuredin cpm) of a T cell line from a diabetic patient to the native B chain(9-23) peptide or to representative peptide analogues containing alaninesubstitutions as indicated. Peripheral blood mononuclear cells wereisolated from diabetic patients and cultured in the presence of insulinB chain (9-23) peptide. After three rounds of restimulation with insulinB chain (9-23), 1×10⁵ T-cells and 7×10⁴ irradiated autologous PBMCs wereadded to each well in a round bottom 96-well plate in complete medium.Cells were cultured for 5 days with analogue, insulin B chain (9-23) ormedium only (BKG), as indicated. On day 4, the cells were pulsed with³H-thymidine and re-cultured for an additional 18 hours. The cultureswere then harvested, counted using liquid scintillation, and the datawas expressed as the mean counts per minute (cpm) of replicatesamples±standard error of the mean (sem).

FIG. 8 is a graph showing the percent of female NOD mice that werediabetic following nine weekly treatments with representative peptideanalogues. Ten mice each were treated subcutaneously beginning on day 24with peptide analogues of the B chain (9-23) containing alaninesubstitutions at residue 12 (open triangles), 13 (squares) or 16 (solidtriangles). All of the mice treated with a control peptide, neurotensin(circles), became diabetic.

FIG. 9 is a graph showing the same data as in FIG. 8, but contrastingonly the A13 analogue-treated group with the control peptide(neurotensin)-treated group.

FIG. 10 is a graph showing the percent of NOD mice that were diabeticfollowing 13 weekly treatments with a representative peptide analogue.Ten mice were treated subcutaneously beginning on day 24 with 400 μg ofneurotensin (squares), B chain (9-23) {diamonds} or a peptide analogueof the B chain (9-23) containing an alanine substitution at residue 13(triangles).

FIG. 11 is a graph showing the effect of representative peptideanalogues on the incidence of diabetes in NOD mice. Four week old femaleNOD mice (n=9) were treated subcutaneously with 20 mg/kg of NBI-6024(A^(16, 19)) at weekly intervals for 12 weeks, followed by every otherweek until 35 weeks of age. The control group (n=10) consisted ofuntreated animals. Mice with blood glucose greater than 200 mg/dL at twoconsecutive time points were considered to be diabetic. The data areexpressed as the percent of non-diabetic over the 35-week study. Thelog-rank test was used to assess whether the results of the twotreatment groups were significantly different. The percent of NOD micethat were diabetic following treatments with NBI-6024 (A^(16, 19)) isindicated at the various time points by squares, and the percent of micethat were diabetic in the control group is indicated by circles.

FIG. 12 is a graph showing the effect of representative peptideanalogues on the incidence of diabetes in NOD mice. Four week old femaleNOD mice (n=13-15) were treated subcutaneously with 20 mg/kg of NBI-6024(A^(16, 19)) or NBI-6201 (a control peptide, neurotensin) at weeklyintervals for 12 weeks, followed by every other week until 35 weeks ofage. An additional control group (n=8) consisted of untreated animals.Mice with blood glucose greater than 200 mg/dL at two consecutive timepoints were considered to be diabetic. The data are expressed as thepercent of non-diabetic over the 35-week study. The log-rank test wasused to assess whether the results of the two treatment groups weresignificantly different. The percent of NOD mice that were diabeticfollowing treatments with NBI-6024 (A^(16, 19)) is indicated at thevarious time points by squares, the percent that were diabetic followingtreatment with the neurotensin peptide is shown by triangles, and thepercent of untreated mice that were diabetic is indicated by circles.

FIGS. 13A-13D are graphs illustrating the immunogenicity ofrepresentative peptide analogues containing B chain residues 9-23 withan alanine substitution at residue 12 (FIG. 13A), residue 13 (FIG. 13B),residue 15 (FIG. 13C) or residue 16 (FIG. 13D). NOD mice were injected2-4 times subcutaneously with the peptide analogue in soluble formbefore assaying the proliferative response of lymph node cells tovarying concentrations of either the peptide analogue or native insulinB chain (9-23) peptide as indicated. Proliferative response was assessedby determining the amount of radioactive thymidine incorporated in thecells (plotted as mean counts per minute (CPM) of triplicate culturewells) by counting in a liquid scintillation counter followingcompletion of the culture period.

FIGS. 14A-14F are graphs showing the immunogenicity of six differentpeptide analogues in the NOD mice. Peptide analogues with two alaninesubstitutions (A12, 13; A12, 15; A12, 16; A13, 15; A13, 16 and A15, 16,as indicated) were injected in NOD mice and after 10 days their lymphnode cells were used in a proliferation assay using differentconcentrations of the immunizing peptide as stimulators. Proliferativeresponse was assessed by determining the amount of radioactive thymidineincorporated in the cells (plotted as mean counts per minute (CPM) oftriplicate culture wells) by counting in a liquid scintillation counterfollowing completion of the culture period.

FIGS. 15A-15D are graphs illustrating the immunogenicity ofrepresentative double substituted peptide analogs of insulin B chain(9-23). The following peptides were tested for their ability to elicitan immune response in NOD mice: A12, 19 (FIG. 15A); A13, 19 (FIG. 15B);A15, 19 (FIG. 15C); A16, 19 (FIG. 15D). Proliferative response as countsper minute of the draining lymph node cells is shown in response to theimmunizing analogue and also to the native insulin B chain (9-23)peptide.

FIG. 16 is a graph showing the ability of a series of triple substitutedpeptides to evoke a T cell proliferative response in NOD mice. Mice wereimmunized separately with representative peptide analogues containingthe following combinations of substitutions: A12, 13, 19; A12, 15, 19;A12, 16, 19; A13, 15, 19; or A13, 16, 19; A15, 16, 19. Lymph node cellswere then used in a proliferation assay, and the response to each of theimmunizing peptides at different concentrations is shown.

FIGS. 17A and 17B are graphs showing the ability of a double substitutedpeptide (A^(16, 19)) to evoke an immune response. Five female NOD micewere immunized subcutaneously with 20 mg/kg of soluble NBI-6024 on days1, 6 and 12. On day 15, the mice were sacrificed, the inguinal lymphnode cells removed and cultured in the presence of varyingconcentrations (0-50 μM) of either NBI-6024 (FIG. 17A) or insulin Bchain (9-23) peptide (FIG. 17B). The extent of T-cell proliferation wasdetermined using ³H-thymidine incorporation. The response is expressedas mean CPM±SEM of triplicate cultures.

FIG. 18 is a histogram showing a comparison of the cytokines produced byimmune cells induced by A^(16, 19) (NBI-6024) peptide in the presence orabsence of adjuvant. Groups of NOD mice were immunized with the NBI-6024alone or emulsified with CFA. The cytokines IL-2 and IL-4, as indicated,were measured at 25 μM of NBI-6024 and expressed as pg/mL aftersubtracting the background values.

DETAILED DESCRIPTION OF THE INVENTION

Prior to setting forth the present invention, it may be helpful to anunderstanding thereof and to provide definitions of certain terms thatare used herein.

“Insulin B chain” refers to a 30 amino acid polypeptide present as oneof the two disulfide-linked polypeptides that make up insulin. Thesequence of human insulin B chain is provided in SEQ ID NO:1, and thesequence of residues 9-23 of human B chain is provided in FIG. 1 and SEQID NO:2.

“Peptide analogues” of the insulin B chain comprise at least 15 aminoacid residues derived from residues 9-23 of human insulin B chain (SEQID NO:2), with at least one difference in amino acid sequence betweenthe analogue and the native B chain. Within a peptide analogue, at leastone difference in amino acid sequence occurs at residue 12, 13, 15and/or 16. In addition, residue 19 may be substituted, and otheralterations are possible. Preferably, a peptide analogue containsbetween 1 and 4 substitutions within residues 9-23, relative to a nativeinsulin B chain (9-23) sequence, although a greater number ofsubstitutions (e.g., 5 or 6) may be possible. Additional residuesderived from insulin B chain may be included, up to the full 30 residuesof native B chain, preferably up to a total of 25 residues, morepreferably up to a total of 16 or 18 residues of the peptide analogue.Within a preferred embodiment, residue 24 of insulin B chain is alsoincluded in the peptide analogue. Sequences that are not derived frominsulin B chain may, but need not, be present at the amino and/orcarboxy terminus of the peptide analogue. Such sequence(s) may be used,for example, to facilitate synthesis, purification or solubilization ofthe peptide analogue.

Unless otherwise indicated, a named amino acid refers to the L-form. AnL-amino acid residue within the native peptide sequence may be alteredto any one of the 20 L-amino acids commonly found in proteins, any oneof the corresponding D-amino acids, rare amino acids, such as4-hydroxyproline or hydroxylysine, or a non-protein amino acid, such asβ-alanine or homoserine. Also included with the scope of the presentinvention are analogues comprising amino acids that have been altered bychemical means such as methylation (e.g., α-methylvaline); amidation ofthe C-terminal amino acid by an alkylamine such as ethylamine,ethanolamine or ethylene diamine; and/or acylation or methylation of anamino acid side chain function (e.g., acylation of the epsilon aminogroup of lysine).

“Residue 12,” “residue 13,” “residue 15,” “residue 16” and “residue 19”(also called position 12, position 13, position 15, position 16 andposition 19, respectively) refer to amino acids 12, 13, 15, 16 and 19 ofinsulin B chain as displayed in FIG. 1. More specifically, the numberingsystem for these residues relates to the amino acid position within thenative human protein, regardless of the length of the peptide analogueor the amino acid position within the analogue. Peptide analogues havingan alanine substitution at residues 12, 13, 15 or 16 are referred to asthe A12, A13, A15 or A16 analogues, respectively.

Peptide Analogues of Insulin B Chain

As noted above, the present invention provides peptide analoguescomprising at least residues 9-23 of human insulin B chain and includingan alteration of the naturally occurring L-valine at position 12,L-glutamate at position 13, L-leucine at position 15 and/or L-tyrosineat position 16, to another amino acid. In one embodiment, peptideanalogues contain additional alterations of one to three L-amino acidsat positions 12, 13, 15, 16 and/or 19 of insulin B chain. Preferably,the peptide analogues contain two or three alterations in which one ofthe substituted residues is at position 19.

The portion of a peptide analogue that is derived from insulin B chainis typically 15-30 residues in length, preferably 15-18 residues inlength, and more preferably 15-16 residues in length. Particularlypreferred peptide analogues contain 15 amino acids derived from insulinB chain.

As noted above, peptide analogues comprising any amino acidalteration(s) at the positions recited above are within the scope ofthis invention. Preferred peptide analogues contain non-conservativesubstitutions (i.e., alterations to amino acids having differences incharge, polarity, hydrophobicity and/or bulkiness). Particularlypreferred analogues contain alterations of one or more residues toalanine.

Peptide analogues may be synthesized by standard chemistry techniques,including automated synthesis. In general, peptide analogues may beprepared by solid-phase peptide synthesis methodology which involvescoupling each protected amino acid residue to a resin support,preferably a 4-methyl-benzhydrylamine resin, by activation withdicyclohexylcarbodiimide to yield a peptide with a C-terminal amide.Alternatively, a chloromethyl resin (Merrifield resin) may be used toyield a peptide with a free carboxylic acid at the C-terminus.Side-chain functional groups may be protected as follows: benzyl forserine and threonine; cyclohexyl for glutamic acid and aspartic acid;tosyl for histidine and arginine; 2-chlorobenzyloxycarbonyl for lysine;and 2-bromobenzyloxycarbonyl for tyrosine. Following coupling, thet-butyloxycarbonyl protecting group on the alpha amino function of theadded amino acid may be removed by treatment with trifluoroacetic acidfollowed by neutralization with di-isopropyl ethylamine. The nextprotected residue is then coupled onto the free amino group, propagatingthe peptide chain. After the last residue has been attached, theprotected peptide-resin is treated with hydrogen fluoride to cleave thepeptide from the resin and deprotect the side chain functional groups.Crude product can be further purified by gel filtration, HPLC, partitionchromatography or ion-exchange chromatography, using well knownprocedures.

Peptide analogues within the present invention (a) should not stimulateNOD mouse T cell clones specific to the native insulin B chain (9-23)peptide (SEQ ID NO:2), or should stimulate such clones at a level thatis lower than the level stimulated by the native peptide; (b) should notstimulate insulin B chain (9-23) specific human T cells from patients;(c) should be immunogenic in the NOD mouse; (d) should reduce theincidence of diabetes in NOD mice and (e) may inhibit a response of Tcell clones specific to the native insulin B chain (9-23) peptide (SEQID NO:2). Thus, candidate peptide analogues may be screened for theirability to treat diabetes by assays measuring T cell proliferation,immunogenicity in NOD mice and the effect on the incidence of thedisease in NOD mice. Certain representative assays for use in evaluatingcandidate peptide analogues are discussed in greater detail below. Thoseanalogs that satisfy the above criteria are useful therapeutics.

Candidate peptide analogues may initially be tested for the ability tostimulate T cells specific to the native insulin B chain (9-23) peptide(SEQ ID NO:2) (from clonal cell lines or isolated from patients). Suchtests may be performed using a direct proliferation assay in whichnative B chain (9-23) reactive T cell lines or T cells isolated frompatients are used as target cells. T cell lines may generally beestablished, using well known techniques, from lymph nodes taken fromrats injected with B chain (9-23). Lymph node cells may be isolated andcultured for 5 to 8 days with B chain (9-23) and IL-2. Viable cells arerecovered and a second round of stimulation may be performed with Bchain (9-23) and irradiated splenocytes as a source of growth factors.After 5 to 6 passages in this manner, the proliferative potential ofeach cell line is determined. To perform a proliferation assay, B chain(9-23)-reactive T cell lines may be cultured for three days with variousconcentrations of peptide analogues and irradiated, autologoussplenocytes. After three days, 0.5-1.0 μCi of [³H]-thymidine is addedfor 12-16 hours. Cultures are then harvested and incorporated countsdetermined. Mean CPM and standard error of the mean are calculated fromtriplicate cultures. Peptide analogues yielding results that are lessthan three standard deviations of the mean response with a comparableconcentration of B chain (9-23) are considered non-stimulatory. Peptideanalogues which do not stimulate proliferation at concentrations of lessthan or equal to 20-50 μM are suitable for further screenings.

Candidate peptides that do not stimulate B chain (9-23) specific Tcells, and preferably inhibit a response of such T cells in vitro, arefurther tested for their immunogenicity in the NOD mouse. Briefly,groups of NOD mice may be immunized with 100-400 μg of the candidatepeptides subcutaneously in mannitol acetate buffer three times within aperiod of 10-15 days. Following the last immunization, lymph node cellsand/or spleen cells may be used in a proliferation assay in whichdifferent concentrations of the immunizing peptide are cultured with thecells for 3-4 days. The last 18 hours of culture may be performed withtritiated thymidine. Cells may then be harvested and counted in ascintillation counter, and the proliferative response may be expressedas CPM±SEM. Candidate peptides that induce a proliferation that is atleast 2-fold higher than the background (no antigen) at 25 μM of thepeptide are considered to be immunogenic. Alternatively, the candidatepeptide analogue is considered immunogenic if it elicits a proliferativeresponse following immunization of the NOD mice in complete Freund'sadjuvant. The draining lymph node cells or spleen cells, when culturedin the presence of the immunizing analogue, should induce aproliferation that is at least 2-fold higher than the background (noantigen) at 25 μM of the peptide.

Candidate peptides that can inhibit proliferation by B chain (9-23) arefurther tested for the ability to reduce the incidence of diabetes inNOD mice. Briefly, peptides may be administered to NOD mice in solubleform or emulsified with, for example, incomplete Freund's adjuvant(IFA). Typically, weekly administration of about 400 μg of peptide issufficient. The incidence of diabetes in the treated mice, as well as inuntreated or control mice, is then evaluated by weekly monitoring ofblood glucose levels. A value of 200 mg/dl or more of blood glucose ontwo consecutive occasions is generally considered indicative of theappearance of diabetes. Peptide analogues should result in astatistically significant decrease in the percent of NOD mice afflictedwith diabetes within a monitoring period of up to about 25 weeks.

As noted above, peptide analogues may also inhibit the response of Bchain (9-23) specific human T cells in vitro. Such inhibition may bemeasured by a competition assay in which candidate peptide analogues aretested for the ability to inhibit T cell proliferation induced by nativeB chain (9-23) (SEQ ID NO:2). In such an assay, antigen presenting cellsare first irradiated and then incubated with the competing peptideanalogue and the native B chain (9-23) peptide. T cells are then addedto the culture. Various concentrations of candidate peptide analoguesare included in cultures which may be incubated for a total of 4 days.Following the incubation period, each culture is pulsed with, forexample, 1 μCi of [³H]-thymidine for an additional 12-18 hours. Culturesmay then be harvested on fiberglass filters and counted as above. MeanCPM and standard error of the mean can be calculated from datadetermined in triplicate cultures. Peptide analogues that reduceproliferation by at least 25% at a concentration 20-50 μM are preferred.

Treatment and Prevention of Diabetes

As noted above, the present invention provides methods for treating andpreventing Type I diabetes by administering to the patient atherapeutically effective amount of a peptide analogue of insulin Bchain as described herein. Diabetic patients suitable for such treatmentmay be identified by criteria accepted in the art for establishing adiagnosis of clinically definite diabetes. Such criteria may include,but are not limited to, low (less than tenth or first percentile ofcontrols) first phase insulin secretion following an intravenous glucosetolerance test (IVGTT) or the persistence of high titer antibodies toislet antigens such as insulin, GAD65 and/or ICA512.

Patients without clinically definite diabetes who may benefit fromprophylactic treatment may generally be identified by any predictivecriteria that are accepted in the art. Patients who are not franklydiabetic may be predicted to develop diabetes in the coming years (1-5yrs) based upon the following criteria: i) family history—first degreerelatives are automatically in the high risk group unless they have aprotective HLA allele; ii) genetic make-up—i.e., the presence or absenceof an HLA allele that is associated with a high risk of diabetes (e.g.,DR3/4; DQ8); iii) presence or absence of high titer autoantibodies intheir blood to any or all of the antigens: insulin, GAD65 and/or ICA512; and iv) intravenous glucose tolerance test (IVGTT): low first-phaseinsulin secretion, usually defined as below the tenth or firstpercentile of normal controls, typically precedes the development oftype I diabetes by 1-5 years. In general, several of the above criteriamay be considered. For example, the chances of developing diabetes in 5years for a first degree relative of an individual with diabetes areestimated to be: 100% for relatives with all 3 autoantibodies listedabove; 44% for relatives with 2 antibodies; 15% for relatives with oneantibody; and 0.5% for relatives with no antibodies. Among 50 firstdegree relatives of patients with Type I diabetes followed to the onsetof diabetes, 49/50 expressed one or more of the above listedautoantibodies.

Effective treatment of diabetes may be determined in several differentways. Satisfying any of the following criteria, or other criteriaaccepted in the art, evidences effective treatment. Criteria mayinclude, but are not limited to, delay in developing frankhyperglycemia, lowered frequency of hyperglycemic events and/orprolongation of normal levels of C-peptide in the blood of the patients.

Peptide analogues of the present invention may be administered eitheralone, or as a pharmaceutical composition. Briefly, pharmaceuticalcompositions of the present invention may comprise one or more of thepeptide analogues described herein, in combination with one or morepharmaceutically or physiologically acceptable carriers, diluents orexcipients. Such compositions may comprise buffers such as neutralbuffered saline, phosphate buffered saline and the like, carbohydratessuch as glucose, mannose, sucrose or dextrans, mannitol, proteins,polypeptides or amino acids such as glycine, antioxidants, chelatingagents such as EDTA or glutathione, adjuvants (e.g., aluminum hydroxide)and preservatives. In addition, pharmaceutical compositions of thepresent invention may also contain one or more additional activeingredients, such as, for example, sustained delivery systems or otherimmunopotentiators.

Compositions of the present invention may be formulated for the mannerof administration indicated, including for example, for oral, nasal,venous, intracranial, intraperitoneal, subcutaneous, or intramuscularadministration. Within other embodiments of the invention, thecompositions described herein may be administered as part of a sustainedrelease implant. Within yet other embodiments, compositions of thepresent invention may be formulated as a lyophilizate, utilizingappropriate excipients which provide stability as a lyophilizate and/orfollowing rehydration.

Pharmaceutical compositions of the present invention may be administeredin a manner appropriate to the disease to be treated (or prevented). Thequantity and frequency of administration will be determined by suchfactors as the condition of the patient, and the type and severity ofthe patient's disease. Within particularly preferred embodiments of theinvention, the peptide analogue may be administered at a dosage rangingfrom 0.1 to 100 mg/kg, although appropriate dosages may be determined byclinical trials. Patients may be monitored for therapeutic effectivenessby delay in progression to frank diabetes and sustained use of insulinfor maintaining normoglycemia as described above.

The following examples are offered by way of illustration and not by wayof limitation.

EXAMPLE 1 Preparation of Peptides

This Example illustrates the synthesis of representative peptideanalogues.

Peptides were synthesized by solid phase methodology on a peptidesynthesizer (Beckman model 990). Peptides with an amidatedcarboxyl-terminus were prepared with a p-methylbenzhydrylamine resin(MBHA resin); for peptides with a free carboxyl-terminus, a Merrifieldresin coupled with the appropriately protected amino acid was used. Bothresins were obtained from Bachem Fine Chemicals (Torrance, Calif.).Derivatized amino acids (Bachem Fine Chemicals) used in the synthesiswere of the L-configuration unless specified otherwise, and theN-alpha-amino function protected exclusively with the t-butyloxycarbonylgroup. Side-chain functional groups were protected as follows: benzylfor serine and threonine; cyclohexyl for glutamic acid and asparticacid; tosyl for histidine and arginine; 2-chlorobenzyloxycarbonyl forlysine and 2-bromobenzyloxycarbonyl for tyrosine. Coupling of thecarboxyl-terminal amino acid to the MBHA resin was carried out withdicyclohexylcarbodiimide and the subsequent amino acids were coupledwith dicyclohexylcarbodiimide according to Ling et al. (Proc. Natl.Acad. Sci. USA 81:4302, 1984). After the last amino acid wasincorporated, the t-butyloxycarbonyl protecting group was removed andthe peptide-resin conjugate treated with a mixture of 14 ml hydrofluoricacid (HF), 1.4 ml anisole, and 0.28 ml methylethyl sulfide per gram ofresin conjugate at −20° C. for 0.5 hr and at 0° C. for 0.5 hr. HF wasremoved in vacuum at 0° C., and the resulting peptide and resin mixturewas washed twice with diethyl ether and twice with chloroform anddiethyl ether alternately. The peptide was extracted five times with 2 Macetic acid, and the extract lyophilized. The lyophilized product wasfirst purified on a column of Sephadex G-25 fine (Pharmacia-LKB,Piscataway, N.J.) developed in 30% acetic acid to remove the truncatedfragments and inorganic salts (Ling et al., 1984). Next, peptides werefurther purified by CM-32 carboxymethylcellulose cation-exchangechromatography (Ling et al., 1984). Final purification was achieved bypartition chromatography on Sephadex G-25 fine (Ling et al., 1984).Alternatively, the crude peptide could be purified by preparative HPLCon a Biotage KP-100 gradient HPLC system. The synthetic product wascharacterized by amino acid analysis, mass spectrometric analysis andreversed-phase HPLC.

EXAMPLE 2 Long-Term T Cell Lines

This Example illustrates the preparation of long-term insulin-specificNOD T cell lines.

Insulin specific NOD T cell lines were established by culturinglymphocytes isolated from islet-infiltrating populations by in vitrostimulation with either porcine insulin at 25 μg/ml and irradiated NODislet cells in the presence of irradiated NOD spleen cells as antigenpresenting cells and cytokines. To obtain the infiltrating lymphocytesthe following procedures were performed (see Wegmann et al., Eur. J.Immunol. 24:1853, 1994): the pancreas from the NOD mouse was digestedwith collagenase and individual islets were isolated manually. Theinfiltrating lymphocytes were then obtained by mild trypsin digestion ofthe islets. The insulin specific T cell lines or clones were propagatedby serial stimulation in the presence of NOD spleen cells, porcineinsulin and lymphokines. Clones were obtained by limiting dilution ofthe B chain (9-23) specific T cell lines in the presence of the antigenpresenting cells and porcine insulin at 25 μg/ml. Wells with a growingpopulation of cells following limiting dilution were expanded inappropriate medium, and after one cycle of growth were tested forreactivity to the B chain (9-23) peptide of insulin by evaluating theproliferative response.

EXAMPLE 3 Effect of Peptide Analogues on Proliferation ofInsulin-Specific NOD T Cell Clones

This Example illustrates the effect of representative peptide analogueson T cell proliferation.

Insulin B chain (9-23) (SEQ ID NO:2) specific mouse (NOD) T cell cloneswere isolated from infiltrated islets as described in Example 2. Peptideanalogues with single alanine substitutions were prepared as describedin Example 1. The effect of each analogue on T cell proliferation wasthen evaluated using an assay performed in 96-well flat bottommicrotiter plates (see Daniel et al., Eur. J. Immunol. 25:1056, 1995).Briefly 25,000 T cell clones along with 1 million irradiated NOD spleencells were cultured in the presence of 50 μg/ml of insulin B chain 9-23peptide or any of the alanine substituted peptides listed below intriplicate sets. The plates were incubated for a total of 72 hours in 7%carbon dioxide atmosphere with a pulse of 1 μCi/well of tritiatedthymidine for the last 6-8 hours of culture. Cells were harvested on aglass fiber filter and the associated radioactivity was counted in aliquid scintillation counter. Results are expressed as mean counts perminute of triplicate wells.

The data obtained from five separate T cell clones showed either a lackof proliferation or significantly reduced proliferation (relative to the9-23 native peptide of insulin B chain; SEQ ID NO:2) in the presence ofthe following alanine substituted analogues: A12, A13, A15, A16, A17,and A18. These data are presented in Tables 1 and 2, below. TABLE 1RESPONSE (CPM) OF INSULIN SPECIFIC NOD T CELL CLONES Modified Native Tcell clone Position Residue Substitution PD6-4.3 PD12-2.40 9 S A 1286142234 10 H A 12507 1409 11 L A 14148 2594 12 V A 8292 671 13 E A 142 51914 A none 15 L A 161 1422 16 Y A 98 539 17 L A 553 19321 18 V A 23444785 19 C A 7678 34212 20 G A 2440 38685 21 E A 91 39087 22 R A 655551722 23 O A 14304 75441 no antigen 163 682 Native 9-23 10463 32221

TABLE 2 RESPONSE (cPM) OF INSULIN SPECIFIC MURINE, NOD T CELL CLONESModified Native T Cell Clone Position Residue Substitution PD12-4.4PD12-4.29 PD12-4.34 9 5 A 1000 18422 259 10 H A 823 15484 356 11 L A 47418416 190 12 V A 1129 15041 194 13 E A 373 891 179 14 A none 15 L A 675809 191 16 Y A 779 636 202 17 L A 332 1460 4360 18 V A 225 1193 721 19 CA 4295 6054 689 20 G A 1323 13736 466 21 E A 7900 4904 773 22 R A 131312635 1555 23 G A 3228 18422 791 no antigen 350 789 231 Native 9-2310000 14820 3614

Table 3 shows the response of four different NOD derived T cell clonesto the double alanine substituted peptide analog A16, A19 (NBI-6024;16Y>A/19C>A). NOD T cell clones were incubated in the presence of 50 μMof either the native B chain (9-23) peptide or NBI-6024. The data inTable 3 represent the mean of triplicate sample±standard error of themean. Within Table 3, S.I. (Stimulation Index)=proliferation (cpm) inthe presence of the peptide/proliferation (cpm) in medium alone. Thesedata show a significant response when the cells were cultured with thenative B chain (9-23) peptide, but little or no proliferation overmedium only (background) in the presence of NBI-6024. TABLE 3PROLIFERATIVE RESPONSE OF INSULIN B CHAIN (9-23) SPECIFIC MURINE T CELLCLONES TO 50 μM OF B CHAIN (9-23) OR THE ANALOGUE A^(16, 19 (NBI-6024))T Cell Exp. Insulin B Chain (9-23) NBI-6024 (A^(16, 19)) Clone No.Medium Only Mean cpm ± sem S.I. cpm ± sem S.I PD12-2.35 1  688 ± 227120,886 ± 7,171 175.7 841 ± 88 1.22 2 493 ± 20 100,521 ± 1,581 203.89 452 ± 179 0.91 PD12-2.40 1 170 ± 8   16,730 ± 3,835 98.4 272 ± 34 1.162 1,834 ± 638   176,359 ± 36,306 96.16 1,863 ± 451  1.01 PD12-4.1 1 215± 17  28,593 ± 4664  132.99 566 ± 30 2.63 PD12-4.9 1  9,111 ± 1,889 45,541 ± 5,222 4.99 12,313 ± 1,372 1.35 2  7,202 ± 2,773  65,624 ±4,979 9.1 6,171 ± 725  0.85 

EXAMPLE 4 Antagonism of T Cell Proliferation Assay

This Example illustrates the inhibition of the response of B chain(9-23) specific mouse T cell clones to the insulin B chain (9-23)peptide by representative peptide analogues.

Peptide analogues of B chain (9-23) containing alanine substitutions atresidue 12, 13, 15 or 16 or the doubly substituted peptide at positions16 and 19 (A^(16, 19); NBI-6024) were prepared as described inExample 1. T cell antagonism was detected by evaluating the ability ofthe peptide analogues to inhibit T cell proliferation induced by nativeB chain (9-23) (SEQ ID NO:2). In this assay, antigen presenting cellswere first irradiated and then incubated with the competing peptideanalogue and the native B chain (9-23) peptide. T cells were then addedto the culture. Various concentrations of candidate peptide analogueswere included in cultures which were incubated for a total of 4 days.Following this incubation period, each culture was pulsed with 1 μCi of[³H]-thymidine for an additional 12-18 hours. Cultures were thenharvested on fiberglass filters and counted as above. Mean CPM andstandard error of the mean were calculated from data determined intriplicate cultures. The results, shown in FIG. 2, indicate that thepeptide analogues containing alanine substitutions at residue 12, 13 or16 are capable of attenuating the response of the pathogenic insulin Bchain (9-23) T cells.

The ability of the doubly substituted peptide to inhibitinsulin-dependent proliferation by T cells is shown in Table 4 and FIG.3. Within Table 4, the control peptide, NBI-5096, is an unrelatedpeptide from myelin basic protein. The percent inhibition was calculatedas: (1-experimental cpm/insulin peptide cpm)×100%. TABLE 4 INHIBITION OFINSULIN B CHAIN (9-23) PEPTIDE RESPONSE IN TWO MURINE NOD T CELL CLONESBY A PEPTIDE ANALOGUE % In- Clone Conditions CPM ± SEM hibitionPD12-2.35 Medium Only   213 ± 17 B(9-23) 5 μM  7,840 ± 528 B(9-23) 5μM + 10 μM NBI-6024  4,441 ± 626 43.0 B(9-23) 5 μM + 50 μM NBI-6024 1,389 ± 218 82.0 B(9-23) 5 μM + 10 μM NBI-5096 10,089 ± 1,113 N/AB(9-23) 5 μM + 50 μM NBI-5096 10,125 ± 887 N/A PD12-2.40 Medium Only  305 ± 13 B(9-23) 5 μM  9,149 ± 1,062 B(9-23) 5 μM + 10 μM NBI-6024 6,379 ± 1,485 30.0 B(9-23) 5 μM + 50 μM NBI-6024  4,305 ± 941 52.9B(9-23) 5 μM + 10 μM NBI-5096 12,336 ± 1,556 N/A B(9-23) 5 μM + 50 μMNBI-5096 17,988 ± 584 N/AN/A = Not applicable as no inhibition was observed.

The ability of NBI-6024 to block the B chain (9-23) peptide-inducedstimulation of NOD derived T clones suggests that the alterations atpositions 16 and 19 of the native insulin B chain (9-23) peptide did notalter the ability of the analogue to be recognized by the pathogenic Tcells. Moreover, these results indicate that the analogue also binds tothe MHC with sufficient affinity to allow for recognition by the insulinB chain (9-23)-specific T cell.

EXAMPLE 5 Effect of Peptide Analogues on Proliferation of T Cell Linesand Clones from Diabetic Patients

This Example illustrates the lack of stimulation of T cell lines andclones derived from diabetic patients by representative peptideanalogues.

Peptide analogues of B chain (9-23) containing alanine substitutions atresidues 13, 15, 16 or 17 or the doubly substituted alanine analogA^(16, 19) (NBI-6024) were prepared as described in Example 1. T celllines from diabetic patients were prepared by isolating lymphocytes fromthe blood of the patient by subjecting the blood to a density gradientseparation. Isolated lymphocytes were then cultured in the presence ofthe insulin B chain (9-23) peptide (10 μM) and recombinant human IL-2 inthe presence of 5-10% of autologous serum and irradiated autologousperipheral blood lymphocytes in culture medium. Four to five days latercells were harvested and the cycle repeated for 2 or 3 times.

Proliferation of the T cell line, in response to the native B chain(9-23) peptide (SEQ ID NO:2) or to the peptide analogs, was measured byculturing 25,000 to 100,000 T cells in the presence of 50,000-200,000irradiated autologous PBLs and different concentrations of the insulin Bchain (9-23) peptide or a peptide analogue in triplicate cultures.Following 4-5 days of culture, including the last 18 hours withradioactive thymidine, cells were harvested and the associatedradioactivity was counted in a liquid scintillation counter. Results areexpressed as mean counts per minute for each of the peptide analoguestested.

The results shown in FIGS. 4-7, indicate that T cell lines and clonesthat proliferate in response to the native insulin B chain (9-23)peptide (SEQ ID NO:2) are not stimulated by the peptide analogues. Theresults from these patients and others are summarized in Table 5. TABLE5 PROLIFERATIVE RESPONSE OF PATIENT PBLs TO NATIVE INSULIN PEPTIDE ORTHE ANALOGUE NBI-6024 Stimulation Index* Insulin B (9-23) NBI-6024Patient Number Patient ID [50 μM] [50 μM] 1 100 9.9 0.9 2 200 5.3 1.2 3400 7.8 1.0 4 500 2.1 0.9 5 600 5.8 1.6 6 700 3.2 1.5 7 900 2.6 0.9 81100 3.7 0.8*Stimulation Index = CPM with antigen/CPM with medium alone (no antigen)

The results clearly demonstrate that cells from diabetic patients thatare responsive to the insulin B chain (9-23) peptide do not respond tothe altered peptide ligand, NBI-6024 which has substitutions at position16 and 19. We have also determined that the APL NBI-6024 binds withsimilar affinity to DQ8 antigens. Thus, the absence of stimulation ofthe diabetic patient's T cells by NBI-6024 is not due to anyincompatibility of the peptide with the presenting MHC molecules, but ismore likely due to altered recognition by the B chain (9-23)-specific Tcells.

EXAMPLE 6 Reduction of Incidence of Diabetes in NOD Mice

This Example illustrates the ability of representative peptide analogsto prevent diabetes in NOD mice.

The NOD mouse spontaneously develops diabetes beginning around 3 monthsof age (Makino et al., in Current Topics in Clinical and ExperimentalAspects of Diabetes Mellitus, Sakamoto et al., eds., p. 25-32 (Elsevier,Amsterdam, 1985)). The disease is preceded by cellular infiltration intothe pancreas of T cells beginning even by one month of age. Solublepeptide analogues of B chain (9-23) containing alanine substitutions atresidues 12, 13 or 16 were administered subcutaneously to NOD mice atweekly intervals. 400 μg of each peptide were administered at eachtreatment to ten animals. Following 9 treatments, the percent of mice ineach treatment group that had become diabetic was evaluated by measuringblood glucose levels using a glucometer at weekly intervals. A readingof more than 200 mg/dl of blood glucose on two consecutive observationswas considered indicative of frank diabetes.

As shown in FIG. 8, treatment with each of the alanine-substitutedanalogues resulted in a marked reduction in the incidence of diabetes.The data for the A13 substituted analogue is also shown in FIG. 9.

In another experiment, B chain (9-23), the A13 substituted analogue orneurotensin (as a control) was administered subcutaneously to NOD miceat weekly intervals. 400 μg of each peptide were administered at eachtreatment to ten animals. Following 13 treatments, the percent of micein each treatment group that had become diabetic was evaluated asdescribed above. As shown in FIG. 10, the B chain (9-23) peptide reducedthe incidence of diabetes. This reduction was more pronounced for theA13 substituted analogue.

To determine the ability of the double substituted peptide A^(16, 19)(NBI-6024) to control the development of diabetes in the NOD mice, thepeptide was administered to animals at an early age. Thus, female mice(n=9, approximately 4 weeks old) were treated subcutaneously with 20mg/kg (400 μg/mouse) of NBI-6024 for twelve weeks and then every otherweek until Week 35. Beginning at 9-10 weeks of age, mice were thenmonitored weekly for hyperglycemia, measuring the blood glucose levels.As a control, a group of 10 female mice was left untreated. The resultsfrom this experiment are shown in FIG. 11. As can be seen, treatmentwith NBI-6024 significantly reduced the incidence of diabetes by about60-70%, compared to the untreated group, (p<0.004).

The observations were then confirmed and extended in a secondexperiment. Here, animals (n=13-15) were treated with either NBI-6024 oran unrelated peptide, neurotensin, NBI-6201 as described above. Anadditional group (n=8) was left untreated. As shown in FIG. 12,treatment with 20 mg/kg of the altered peptide NBI-6024 resulted in areduced incidence of diabetes compared to either the neurotensin treatedor untreated group.

These results demonstrate that the altered peptide ligand NBI-6024,designed around insulin B chain (9-23) peptide was capable of conferringprotection to animals at risk to develop diabetes spontaneously. It islikely that T cells that recognize other pancreas antigens are presentin these animals, yet they too appear to be regulated by the insulinAPL. The timing of administration was approximately at the same timethat autoreactive lymphocytes begin to infiltrate the pancreas andinitiate the destructive process. These results offer hope that earlyintervention with this APL may prove useful in delaying or preventingthe onset of Type I diabetes in people.

EXAMPLE 7 Immunogenicity of Representative Peptide Analogues

This Example illustrates the immunogenicity of representative peptideanalogues in NOD mice.

Groups of 34 NOD mice were immunized with 100-400 μg of peptideanalogues subcutaneously in mannitol acetate buffer three times within aperiod of 10-15 days. Following the last immunization, lymph node cellsand/or spleen cells were used in a proliferation assay in whichdifferent concentrations of the immunizing peptide were cultured withthe cells for 34 days. The last 18 hours of culture included tritiatedthymidine. Cells were harvested and counted in a scintillation counterand the response is expressed as CPM±SEM. These results, shown in FIG.13-16, indicate that these representative peptide analogues have theability to bind to the mouse MHC molecules and be recognized by thecorresponding T cells.

The ability of the double substituted peptide NBI-6024 (A^(16, 19)) toinduce a cellular immune response in NOD mouse strain was nextdetermined. Two female NOD mice were immunized with 10 mg/kg NBI-6024either as an aqueous suspension or, as a control, emulsified in completeFreund's adjuvant (CFA). On Day 8, three days following the lastinjection, the mice were sacrificed, the spleen and inguinal lymph nodecells were removed and pooled, and a single-cell suspension wasprepared. Cells were cultured in the presence of varying concentrations(0-25 μM) of NBI-6024. The ability of these lymphoid cells toproliferate in response to NBI-6024 was measured in vitro by[³H]-thymidine incorporation.

The results are presented in Table 6, in which the response is expressedas mean CPM±SEM of triplicate cultures. Lymph node cells isolated frommice immunized with the analogue in CFA showed a strong proliferativeresponse to challenge with the immunizing analogue in a dose-dependentmanner (Table 6). These results indicate that alterations made in thenative insulin B chain (9-23) sequence at positions 16 and 19 have notaffected the ability of the peptide to bind the NOD disease-associatedMHC haplotype molecule and, more importantly, did not hinder recognitionby T cells. TABLE 6 PROLIFERATIVE RESPONSE OF LYMPH NODE CELLS TONBI-6024 FROM NOD MICE IMMUNIZED WITH NBI-6024 IN CFA NBI-6024Proliferative Response (μM) (CPM ± SEM) 0  2,445 ± 137   1 140,061 ±7,289 5 187,711 ± 2,548 25 218,149 ± 4,462

In addition, both the spleen and inguinal lymph nodes cells isolatedfrom soluble administered peptide exhibited a strong proliferativeresponse to the APL when challenged in vitro with NBI-6024 (Table 7 andFIGS. 17A and 17B). Even more impressive was the finding thatNBI-6024-derived lymphocytes from mice immunized with the solublepeptide also responded to Insulin B chain (9-23). This cross-reactivefeature was not seen with CFA-emulsified peptide. This ability of thesoluble peptide to induce a cross-reactive response may be desirable incontrolling diabetes, as it may help to mobilize the protectiveNBI-6024-specific T cells to the pathogenic target tissue. TABLE 7PROLIFERATIVE RESPONSE TO NBI-6024 OR NATIVE INSULIN B-CHAIN (9-23) OF TCELLS FROM NOD MICE IMMUNIZED WITH SOLUBLE NBI-6024 Peptide NBI-6024Induced Conc. CPM ± SEM CPM ± SEM Cell Line [μM] NBI-6024 Insulin B(9-23) Mouse #1 0  19,130 ± 2191  19,130 ± 2191 1  48,319 ± 1918  16,870± 4469 5 160,673 ± 2269  21,292 ± 3216 25 268,005 ± 11198  33,317 ± 3619Mouse #2 0  21,588 ± 2326  21,588 ± 2326 1  54,519 ± 5666  17,262 ± 6025 126,123 ± 13851  19,648 ± 2169 25 202,707 ± 8125  30,612 ± 3557 Mouse#3 0  24,006 ± 2803  24,006 ± 2803 1  64,239 ± 9493  25,825 ± 3841 5140,836 ± 11778  58,567 ± 2737 25 240,278 ± 15015 113,366 ± 515

To determine the type of T cells produced following solubleadministration of NBI-6024, the culture supernatants from immunelymphoid cells were removed 48 hours following the initiation of cultureand the levels of various cytokines measured using standard ELISAtechnology. Strikingly, the cytokine production profile of T cells frommice immunized with soluble NBI-6024 produced the Th2 cytokines,interleukin-4 (FIG. 18) and interleukin-5 (Table 8), and not theTh1-derived interleukin-2. Within Table 8, values are expressed in pg/mlas mean of triplicates±SEM. As a control, NBI-6024 emulsified with CFAdid induce the expected Th1-cytokine profile (IL-2) from immune T cellsupon in vitro stimulation. TABLE 8 CYTOKINE RESPONSE OF SOLUBLE NBI-6024INDUCED T CELLS CULTURED WITH NBI-6024 NBI-6024 Cytokine (pg/ml) [mM]IL-2 IL-4 IL-5 0 <15 pg/ml 134 ± 0 1,814 ± 332 1 <15 pg/ml  684 ± 929,999 ± 503 5 <15 pg/ml 1,653 ± 51  23,496 ± 684  25 <15 pg/ml 2,102 ±85  28,062 ± 141 

The ability of the soluble subcutaneous administration of NBI-6024 toinduce Th2-like cells is a desirable feature, as such cells areassociated with recovery from diabetes and other organ-specificautoimmune diseases (Sarvetnick, J. Exp. Med. 184:1597-1600, 1996; Shawet al., 1997; Balasa et al., J. Exp. Med. 186:385-391, 1997). TheseTh2-derived cytokines have strong anti-inflammatory activities thatsuppress development of pro-inflammatory cytokine-secretingauto-reactive Th1 cells that mediate disease.

All of the above U.S. patents, U.S. patent application publications,U.S. patent applications, foreign patents, foreign patent applicationsand non-patent publications referred to in this specification and/orlisted in the Application Data Sheet, are incorporated herein byreference, in their entirety.

From the foregoing, it will be evident that although specificembodiments of the invention have been described herein for the purposeof illustrating the invention, various modifications may be made withoutdeviating from the spirit and scope of the invention. Accordingly, thepresent invention is not limited except as by the appended claims.

1-27. (canceled)
 28. A peptide analogue comprising residues 9 to 23 ofhuman insulin B chain, wherein the peptide analogue differs in sequencefrom native human insulin B chain residues 9 to 23 due to substitutionsat between 1 and 4 amino acid positions, and wherein one substitutionoccurs at residue
 19. 29. The peptide analogue of claim 28, wherein thepeptide analogue differs in sequence from native human insulin B chainresidues 9 to 23 due to substitutions at between 2 and 4 amino acidpositions, and wherein at least one substitution occurs at a residueselected from the group consisting of residues 12, 13, 15 and
 16. 30.The peptide analogue of claim 29, wherein the peptide analogue differsin sequence from native human insulin B chain residues 9 to 23 due tosubstitutions at 4 amino acid positions, and wherein at least onesubstitution occurs at a residue selected from the group consisting ofresidues 12, 13, 15 and
 16. 31. The peptide analogue of claim 29,wherein the peptide analogue differs in sequence from native humaninsulin B chain residues 9 to 23 due to substitutions at 3 amino acidpositions, and wherein at least one substitution occurs at a residueselected from the group consisting of residues 12, 13, 15 and
 16. 32.The peptide analogue of claim 29, wherein the peptide analogue differsin sequence from native human insulin B chain residues 9 to 23 due tosubstitutions at 2 amino acid positions, and wherein one substitutionoccurs at a residue selected from the group consisting of residues 12,13, 15 and
 16. 33. The peptide analogue of claim 32, wherein the peptideanalogue differs in sequence from native human insulin B chain residues9 to 23 due to substitutions at residues 12 and
 19. 34. The peptideanalogue of claim 32, wherein the peptide analogue differs in sequencefrom native human insulin B chain residues 9 to 23 due to substitutionsat residues 13 and
 19. 35. The peptide analogue of claim 32, wherein thepeptide analogue differs in sequence from native human insulin B chainresidues 9 to 23 due to substitutions at residues 15 and
 19. 36. Thepeptide analogue of claim 32, wherein the peptide analogue differs insequence from native human insulin B chain residues 9 to 23 due tosubstitutions at residues 16 and
 19. 37. The peptide analogue of claim28, wherein residue 19 is an alanine residue.
 38. The peptide analogueof claim 29, wherein residue 19 is an alanine residue.
 39. The peptideanalogue of claim 32, wherein residue 19 is an alanine residue.
 40. Thepeptide analogue of claim 36, wherein residue 19 is an alanine residue.41. The peptide analogue of claim 36, wherein residue 16 is an alanineresidue.
 42. The peptide analogue of claim 32, wherein the peptideanalogue comprises no more than 18 residues of human insulin B chain.43. The peptide analogue of claim 32, wherein the peptide analoguecomprises no more than 16 residues of human insulin B chain.
 44. Thepeptide analogue of claim 32, wherein the peptide analogue comprises nomore than 15 residues of human insulin B chain.
 45. A pharmaceuticalcomposition comprising a peptide analogue according to claim 32 incombination with a physiologically acceptable carrier or diluent.
 46. Apharmaceutical composition comprising a peptide analogue according toclaim 36 in combination with a physiologically acceptable carrier ordiluent.
 47. A pharmaceutical composition comprising a peptide analogueaccording to claim 40 in combination with a physiologically acceptablecarrier or diluent.